Oral compositions containing dextranase

ABSTRACT

Oral compositions containing dextranase and a stabilizer/activator selected from the group consisting of manganeous ions, calcium ions, and mixtures thereof.

This application is a continuation-in-part of Ser. No. 419,343,abandoned, filed Nov. 27, 1973, which is a continuation-in-part of Ser.No. 313,304, filed Dec. 8, 1972, now abandoned.

This invention relates to oral compositions containing dextranase and totheir method of preparation. The oral compositions of this invention arecharacterized by their stability and increased activity. This stabilityand increased activity are due to the presence of specific metal ions.

Dextranase has been proposed to be an effective caries-preventive agentuseful in the removal of bacteria on dextran-containing plaque. One ofthe major deterrents to the use of dextranase in oral compositions hasbeen the inability to formulate a liquid carrier which would maintainthe enzyme in an active state. Unfortunately, when dextranase isincorporated into aqueous solution, enzyme inactivation occurs anddextranase is broken down and thereby deactivated to such a degree thatits effectiveness is lost.

Accordingly, it is an object of this invention to provide a stabilizedoral composition containing dextranase in a liquid vehicle.

It is another advantage of this invention to provide an oral compositioncontaining dextranase which has increased activity.

Other advantages of this invention will be apparent from considerationof the following description.

In accordance with certain of its aspects, this invention relates tooral preparations comprising a liquid vehicle, dextranase, and astabilizer/activator selected from the group consisting of manganeousions, calcium ions, magnesium ions, and mixtures thereof.

The oral compositions of this invention are typically a toothpaste, thatis, a dental cream or gel. Such a cream or gel may be employed as atoothpaste or mouth rinse. Generally the compositions also contain apolishing agent, but if the composition is to be used specifically as amouth rinse, then the polishing agent may be omitted.

Dextranase enzymes are produced from a variety of sources all of whichare useful in the present invention. Dextranase enzymes are commonlyproduced by growing Penicillium funiculosium or other fungal sources ina dextran-containing medium. The dextran is commonly a commercial gradeobtained from Leuconostoc mesenterioides. This commercial grade ofdextran contains about 95-percentα-1,6-glucoside linkages and about5-percentα-1,3-glucoside linkages. The Penicillium organism produces thedextranase which particularly hydrolyzes the 1,6-linkages.

Dextranase may also be prepared in accordance with procedures which aredescribed in the art. These include the procedure described by Bowen,"British Dental Journal", Vol. 124, No. 8, dated Apr. 16, 1968, pages343-349. A further procedure is described in U.S. Pat. No. 2,742,399 toTsuchiya et al. (Note also Tsuchiya et al., "Journal of Bacteriology",Vol. 64, page 513).

In the procedure of Bowen, dextran may be prepared from noncariogenicstreptococcal strains such as ATCC 10558, 903-1600, IIA2+3, orLeuconostoc mesenterioides and purified according to the methoddescribed by Wood et al., "Archives of Oral Biology", Vol. 11, 1066,pages 1039 et seq., except that L. mesenterioides is grown at 25° C.

Dextranase may be prepared from dextran by inoculating Penicilliumfunicolosum into flasks containing 250 ml. of a medium containing0.5-percent yeast extract and 1-percent dextran. The flasks areincubated at 30° C. on a shaking incubator for 4 days. The culture inthen centrifuged at 3,000 g. for 20 minutes and filtered through Whatman42 filter paper. Dialysis in 16 mm. "Visking" tubing against deionizedwater and concentrating fifty fold by dialysis against polyethyleneglycol (molecular weight 20,000) follows. The dextranase produced inaccordance with this procedure has a molecular weight of about 200,000to 275,000. If desired, the dextranase may be further purified byfractionation with ammonium sulfate.

Additional procedures for preparing dextranase include that described inU.S. Pat. No. 2,742,399 to Tsuchiya et al.

Dextranases of bacterial origin are also useful the presentcompositions. Bacterial-origin dextranase may be prepared in the generalmanner in which enzymes are derived from bacteria. However, thepreferred source of dextranase for the purposes of this invention is amutan of Bacillus coaguluns, NRRL B-3977 (Beckman dextranase catalogueNo. 680000). Bacterial-origin dextranase may be obtained by the additionof α-1,3-dextran or a mixture of α-1,6-, α-1,3-, and α-1,4-dextrans. Thebacterial strain may be innoculated into a shaker flask or fermentatorfor a period of 1 to 5 days at 25° to 40° C. The sterile growth mediascan consist of the aforementioned dextran or mutan combined with amixture of carbohydrate (starch, glucose, sucrose, cellulose),nitrogeneous compounds (protein digest, gelatin, casein, ammoniumsalts), growth stimulators, (yeast extract, corn steep liquor,distiller's solubles), or minerals. Preservatives may be added and theenzyme decanted, filtered, or centrifuged to precipitate the cells(intracellular dextranase). The extracellular dextranase can beprecipitated with ammonium sulfate, acetone, sodium sulfate, or asimilar salt. The intracellular dextranases are autolyzed and extracted.Following the salt fractionation step, the enzyme can be furtherpurified by a variety of column (DEAE, Sephodex, ECTEOLA,hydroxyapatite) chromatography methods and frozen or stabilized by theaddition of protein, dextran, salt, etc. (The purification steps areusually conducted at refrigerated temperatures.)

The amount of dextranase employed in the oral compositions of theinvention is at least such amount as is effective in promoting oralhygiene. This amount is dependent upon the activity of the dextranasewhich may typically range from 20 to400 units/mg. protein (proteindetermined by the Lowry method) and therefore upon the mode of itspreparation. A typically prepared dextranase enzyme material has anactivity of about 117 units/mg. protein. One Beckman dextranase unit isthe amount of enzyme which produces 1.0 M of reducing sugar from"native" dextran per minute at 35° C. and pH 6.0.

While smaller amounts of dextranase may be used, dextranase having anactivity of about 20 to 400 units/mg. protein may be present in amountsof about 0.001 to 5 percent by weight of the oral composition, whiledextranase having an activity of about 90 or 100 to 150 units/mg proteinmay be preferably present in amounts of about 0.01 to 0.2 percent byweight.

The stabilizer/activator of the instant invention is a metal ion such asmanganese, calcium, magnesium, and mixtures thereof. These ions may beprovided by any suitable source. Typical sources include the nontoxicwater-soluble salts, e.g. chlorides, sulfates, and nitrates. The mostpreferred sources being manganeous chloride, calcium chloride, andmagnesium chloride.

The stabilizer/activator metal ion is typically employed in an amount ofabout 0.001 to about 0.3 percent by weight, preferably about 0.1 to 0.3percent and most preferably about 0.2 to 0.25 percent of the oralcomposition, unless the composition in a mouth rinse concentrate havingan amount generally about 4 times as great to provide for a 3 to 1dilution.

For optimum conditions, the stabilizer/activator is added to thedextranase, and the combination is incubated for 1 hour at 37° C. beforeaddition to the formulation. The dextranase activity can be measured bya number of art recognized assay methods, but for our purposes thereducing sugar assay is used (Noeling and Bernfield, Helv. Chim. Acta.,Vol. 31, page 286, 1948).

The desirable character of the oral compositions of this invention isattained by proportioning cosmetically acceptable and nontoxic aqueousliquid ingredients with solid ingredients to produce an acceptablecarrier material. In general, the liquids in the composition willcomprise chiefly water, glycerine, aqueous solutions of sorbitol,propylene glycol, polyethylene glycol 400, etc., and suitable mixturesthereof. It is advantageous usually to use a mixture of both water andhumectant such as glycerine or sorbitol. The total liquid vehiclecontent will generally be about 20 to 75 percent by weight of theformulation of which water may constitute up to 20 percent, preferablyup to 15 percent, of the formulation.

Toothpaste compositions of this invention form creamy or gel masses ofdesired consistency which are easily extrudable from a pressurizedcontainer or a collapsible aluminum or lead tube.

It is generally preferred to use a binding agent in toothpaste. However,certain synthetic binding agents are incompatible with certain types ofdextranases. If the dextranase used is of a fungal origin, the preferredbinding agents are Irish moss and gum tragacanth. However, if thedextranase used is of a bacterial origin, then the preferred bindingagents are carboxymethylcellulose, hydroxypropylcellulose, andhydroxyethylcellulose. Other conventional binding agents are alsocompatible with bacterial-origin dextranase, for example, Irish moss,gum tragacanth, and gum karaya.

The binder materials is typically employed in amount of up to about 10%by weight, preferably up to about 5% and most preferably about 0.2-1.5%of the oral composition.

Preferably the oral composition is a dental cream or gel and contains asuitable substantially water-insoluble polishing agent which iscompatible with the formulation. Particularly compatible materialsinclude, for example, dicalcium phosphate dihydrate, dicalcium phosphateanhydrous, tricalcium phosphate, magnesium carbonate, calcium sulfate,bentonite, etc., and suitable mixtures thereof. Abrasive resinoussubstances such as the condensation products of melamine and urea withformaldehyde can also be used. It is preferred to use dicalciumphosphate dihydrate, dicalcium phosphate anhydrous, and calciumcarbonate. The polishing agent content is variable, but will generallybe up to about 75 percent by weight of the total formulation, typicallyabout 20 to 75 percent.

It is often desirable to include a compatible organic surface-activeagent to achieve increased prophylactic action, assist in achievingthorough and complete dispersion of the instant composition throughoutthe oral cavity, and render the instant compositions more cosmeticallyacceptable. However, many surface-active agents have been found todeactivate dextranase. One particular class of detergents has been foundto be particularly compatible with dextranase is the N-substituted loweralkyl C₁₂ --C₁₈ fatty acid sulfoacetamides. The most preferredsurface-active agent of this class is N-2-ethyl laurate potassiumsulfoacetamide, ##STR1##

This class of surface-active agents has been found to be compatible withdextranase. In addition to the aforementioned detergent, the sodium,potassium, and ethanolamine salts of N-lauroyl, N-myristoyl, andN-palmitoyl sarcosinates are also compatible with dextranase.

Other suitable surface-active agents include compatible nonionicsurface-active agents, such as condensates of sorbitan monostearate withapproximately 60 moles of ethylene oxide, and condensates of ethyleneoxide with propylene oxide condensates of propylene glycol("Pluronics").

It is preferred to use from about 0.05 to 5 percent by weight of theforegoing surface-active agents in the instant compositions.

Various other materials may also be incorporated into the carrier.Examples thereof are coloring or whitening agents (for example, titaniumdioxide), preservatives (for example, sodium benzoate), silicones,chlorophyll compounds, ammoniated materials such as urea, diammoniumphosphate, and mixtures thereof, alcohol, menthol, and otherconstituents. These adjuvants are incorporated into the instantcompositions in amounts which do not substantially adversely affect theproperties and characteristics of the compositions. These adjuvants aresuitably selected and used in proper amounts depending upon theparticular type of preparation involved.

Additionally, compatible antibacterial agents may be incorporated in thecompositions of the invention. Conventional agents include:

N¹ -(4-chlorobenzyl)-N⁵ -(2,4-dichlorobenzyl)biguanide;

p-chlorophenyl biguanide;

4-chlorobenzylhydryl biguanide;

4-chlorobenzhydrylguanylurea;

N-3-lauroxypropyl-N⁵ -p-chlorobenzylbiguanide;

1,6-di-p-chlorophenylbiguanidohexane;

1-(lauryldimethylammonium)-8-(p-chlorobenzyldimethylammonium)octanedichloride;

5,6-dichloro-2-guanidinobenzimidazole;

N¹ -p-chlorophenyl-N⁵ -laurylbiguanide;

5-amino-1,3-bis(2-ethylhexyl)-5-methylhexahydropyrimidine;

2,2'dihydroxy-3,5,6,3',5',6'hexachlorodiphenylmethane;

2,2'dihydroxy-5,5'dichlorodiphenylmethane;

and their nontoxic acid addition salts. These agents may be employed inamounts ranging from 0.01 to 5 percent and preferably 0.05 to 1.0percent.

Any suitable flavoring or sweetening agent may be employed informulating a flavor for the compositions of the present invention.Examples of suitable flavoring constituents include the flavoring oils,for example, oils of spearmint, peppermint, wintergreen, sassafras,clove, sage, eucalyptus, marjoram, cinnamon, lemon, and orange, as wellas methyl salicylate. Suitable sweetening agents include sucrose,lactose, maltose, sorbitol, sodium cyclamate, and saccharine. Suitably,flavor and sweetening agent may together comprise from about 0.01 to 5percent or more of the compositions of the instant invention.

The oral compositions of the invention may also desirably contain afluorine-providing compound.

Examples of suitable fluorine-providing compounds include sodiumfluoride, stannous fluoride, potassium fluoride, potassium stannousfluoride (SnF₂ KF), sodium hexafluorostannate, stannous chlorofluoride,sodium fluorozirconate, and sodium monofluorophosphate. These materials,which dissociate or release fluorine containing ions in water, suitablyare present in an effective but nontoxic amount, usually within therange to provide about 0.01 to 1 percent by weight of fluorine,preferably about 0.1 percent.

The instant compositions normally have a pH between about 6.0 and 8.0and preferably about 6.0. Suitably a buffering system may be employed toassure maintenance of a pH within the aforesaid range.

In order to determine the increase in dextranase activity due to thepresence of the metal ions of the instant invention, the followingexperiment was conducted in which a dextranase control is compared withmixtures of dextranase and the various metal ions.

To prepare the control 0.5 ml. of a 0.067M phosphate buffer (pH 6.0)which contains 5μg. dextranase per 10μl. phosphate buffer is admixedwith 1.5 mls. phosphate buffer and 2 mls. 2-percent Leuconostocmesenterioides dextran. The control is incubated at 40° C. for 45minutes. In preparing the mixtures containing the metal ions the amountof phosphate buffer is adjusted so that the final volume of all mixturesis 4 mls. The dextranase activity is measured by the reducing sugarassay (Noeling and Bernfield).

The results of these experiments are seen in the following table:

    __________________________________________________________________________                 Ratio Dextranase to                                                                      Amount of 0.067M                                                                        Amount of    Amount of Additional                                                          0.067                          Compositions Phosphate Buffer                                                                         Phosphate Buffer                                                                        Metal Ion    M Phosphate                    __________________________________________________________________________                                                   Buffer                         Dextranase (Control)                                                                       5μg/10μ1                                                                           0.5 ml.    --             1.5 mls.                    Dextranase + Ca.sup.+.sup.+ Dextranase + Mg.sup.+.sup.+ Dextranase +          Mn.sup.+.sup.+ Dextranase + Ca.sup.+.sup.++Mn.sup.+.sup.+ Dextranase +        Ca.sup.+.sup.++Mg.sup.+.sup.+ Dextranase + Mn.sup.+.sup.++Mg.sup.+.sup.+      Dextranase + Ca.sup.+.sup.++Mg.sup.+.sup.++ Mn.sup.+.sup.+                                  ##STR2##                                                                                 ##STR3##                                                                                ##STR4##       1.475 mls. 1.475 mls.                                                         1.475 mls. 1.45 mls.                                                          1.45 mls. 1.45 mls.                                                           1.425 mls.                  [CaCl.sub.2 ] = 2 × 10.sup.-.sup.2 M = 29.4 mg./10ml. PO.sub.4          buffer                                                                        [MnCl.sub.2 ] = 2 × 10.sup.-.sup.2 M = 39.6 mg./10ml. PO.sub.4          buffer                                                                        [MgCl.sub.2 ] = 2 × 10.sup.-.sup.2 M = 40.6 mg./10ml. PO.sub.4          buffer                                                                        __________________________________________________________________________     Dextranase is Beckman Corporation Dextranase (Catalogue No. 680000,           activity 117 units/mg. protein)                                               DNSA is dinitrosalicyclic acid                                                2% Dextran = 2% Leuconostoc mesenterioides Dextran (M.W. = 5 - 40 ×     10.sup.6)                                                                

    __________________________________________________________________________    Amount of          Amount       Amount                                                                             Percent Activity                         2-% Dextran        DNSA         Water                                                                              Relative to Control                      __________________________________________________________________________    2.0 mls.           2.0 mls.     25 mls.                                                                              100%                                    ##STR5##                                                                              Add Enzyme Here Incubate at 40° C. for 45                                         ##STR6##                                                                            Mix and Δ to boiling; Cool in                                                  ##STR7##                                                                            745% 584% 1100% 1375% 480%             __________________________________________________________________________                                           1220%                              

As can be seen from the table the presence of the metal ion increasesthe dextranase activity by quite significant amounts of from about 5 to14 times that of the control.

In order to determine the stabilizing effect of these metal ions ondextranase, the following toothpaste compositions were formulated andtested over a 9-week aging period.

    __________________________________________________________________________                     EXAMPLE 1  EXAMPLE 2                                         Components       Percent by Weight                                                                        Percent by Weight                                 __________________________________________________________________________    Glycerine        28.37      28.37                                             Sodium saccharin 0.20       0.20                                              Sodium benzoate  0.50       0.50                                              Carboxymethylcellulose                                                                         0.80       0.80                                              Water (Deionized)                                                                              15.38      15.18                                             Calcium carbonate                                                                              5.00       5.00                                              Dicalcium phosphate dihydrate                                                                  46.75      46.75                                             N-2-ethyl laurate potassium                                                     sulfoacetamide 2.00       2.00                                              Flavor           0.80       0.80                                              Detranase (Beckman, activity                                                    117 units/mg. protein)                                                                       0.20       0.20                                              Manganese chloride tetrahydrate                                                                --         0.20                                              __________________________________________________________________________

The results are as follows:

    ______________________________________                                                 Example 1    Example 2                                               ______________________________________                                        0 weeks    100%           100%                                                3 weeks    85%            135%                                                6 weeks    15%             97%                                                9 weeks     9%             86%                                                ______________________________________                                    

The toothpaste composition containing 0.20-percent manganese chloride isabout 9.5 times more stable than the corresponding toothpaste having nomanganese ions present. Calcium and magnesium ions also stabilize thetoothpaste.

The above experiments clearly show the impressive stabilizing andactivating characteristics of the metal ions herein claimed.

The following specific examples are further illustrative of the natureof the present invention, but it is understood that the invention is notlimited thereto.

EXAMPLE 3

The following oral gel is prepared:

    ______________________________________                                                          Percent by Weight                                           Glycerine           10.00                                                     Sodium saccharin    0.20                                                      Sodium benzoate     0.50                                                      Irish moss          2.00                                                      Sorbitol            74.24                                                     Sodium N-lauroyl sarcosinate                                                                      1.50                                                      Ethanol             10.00                                                     Dextranase (Fungal origin,                                                     200 units/mg. protein)                                                                           0.01                                                      MnCl.sub.2.4H.sub.2 O                                                                             0.25                                                      ______________________________________                                    

EXAMPLE 4

The following mouthrinse concentrate is prepared:

    ______________________________________                                                           Percent by Weight                                          Glycerine            45.0                                                     Ethanol              40.0                                                     Water (Deionized)    8.8                                                      Tween 80             2.0                                                      Sodium saccharin     2.0                                                      Dextranase (Beckman, activity                                                  117 units/mg. protein)                                                                            2.0                                                      MnCl.sub.2.4H.sub.2 O                                                                              0.8                                                      Flavor               0.3                                                      Dye                  0.3                                                      ______________________________________                                    

Upon use the above concentrate is diluted 1 part mouthrinse concentrateto 3 parts water.

It will be apparent that various modifications may be made in theexamples which fall within the scope of the invention.

What is claimed is:
 1. A dental cream or gel comprising a carriermaterial, dextranase having an activity of about 20 to 400 units/mg.protein in amount of about 0.001 to about 5% by weight and a non-toxiccompound which provides an effective amount of at least 0.001% ofstabilizer/activator manganous metal ions said metal ions being providedfrom water-soluble salts selected from the group consisting ofchlorides, sulfates and nitrates, namely manganous chloride, manganoussulfate and manganous nitrate, said metal ions being effective tostabilize said dextranase and activate its dextranase activity.
 2. Thedental cream or gel of claim 1 in which the dextranase has an activityof about 90 to about 400 units/mg. protein.
 3. The dental cream or gelof claim 2 wherein said carrier material includes about 20 to about 75%by weight of a liquid vehicle and about 20 to about 75% by weight of apolishing agent.
 4. The dental cream or gel of claim 1 wherein saidmetal ions are provided from chloride salts.